Iron is an element used by eukaryotic organisms and most microorganisms as a cofactor of numerous proteins or enzymes for respiration, DNA synthesis, and many other critical metabolic processes (Baker, et al., Proc Natl Acad Sci USA 100: 3579-3583 (2003)). Cellular iron deficiency can arrest cell proliferation and even cause cell death, whereas the excessive iron will be toxic to cells by reacting with oxygen via the Fenton reaction to produce highly reactive hydroxyl radicals that cause oxidative damage to cells (Baker, et al., Proc Natl Acad Sci USA 100: 3579-3583 (2003); Hentze, M. U., et al., Cell 117: 285-97(2004)). To overcome the dual challenges of iron deficiency and overload, a family of iron carrier glycoproteins collectively called transferrins has evolved in nearly all organisms to tightly control cellular iron uptake, storage, and transport to maintain cellular iron homeostasis (Williams, J., Trends Biochem. Soc. 7: 394-397 (1982)). The transferrin protein family includes several homologous glycoproteins generally having a molecular weight of approximately 80 kDa and an ability to bind iron, and is divided into four subsets: (1) serum transferrins (TF) which have a role in iron transport in the body; (2) lactoferrins (LF) found in mammalian extracellular secretions such as milk, tears, pancreatic fluid and other bodily secretions of mammals; (3) melanotransferrins (mTF) which is present on the surface of melanocytes and in liver and intestinal epithelium; and (4) ovotransferrins (oTF) found in bird and reptile oviduct secretions and egg white. While all members of the transferrin protein family can bind iron to control free iron level, human serum transferrin provides both a means of transporting iron from the sites of absorption and storage to the sites of utilization, as well as protection against the damaging effects of iron-catalyzed free radicals. To date, only TF has been proven to be able to transport iron to cells (Baker, et al., Proc Natl Acad Sci USA 100: 3579-3583 (2003)).
One exemplary TF is a single-chain glycoprotein of 679 amino acid residues including 38 cysteine residues which are all disulfide bonded. TF consists of two homologous halves, each comprising about 340 amino acid residues and sharing about 40% sequence identity (Baker, et al., Proc Natl Acad Sci USA 100: 3579-3583 (2003); Hirose, Biosci. Biotechnol. Biochem. 64:1328-1336 (2000); J. Wally, et al., Biometals 20: 249-62 (2007)). The two homologous halves are shown by X-ray crystallography to fold into two distinct globular lobes called N- and C-terminal lobes (Baker, et al., Proc Natl Acad Sci USA 100: 3579-3583 (2003); Hirose, Biosci. Biotechnol. Biochem. 64:1328-1336 (2000)). Each lobe comprises two dissimilar domains (N1 and N2 in the N-lobe; C1 and C2 in the C-lobe) separated by a deep cleft, where the iron binding site is located. The iron-binding ligands in each lobe are identical, which involves the side chains of an aspartic acid, two tyrosines, a histidine and two oxygen molecules from a synergistic carbonate anion (Baker, et al., Proc Natl Acad Sci USA 100: 3579-3583 (2003); Hentze, M. U., et al., Cell 117: 285-97(2004); Hirose, Biosci. Biotechnol. Biochem. 64:1328-1336 (2000); J. Wally, et al., Biometals 20: 249-62 (2007); Q.-Y. He, et al., “Molecular aspects of release of iron from transferrin,” in: D. M. Templeton, (Ed.), Molecular and Cellular Iron Transport, CRC Press, 2002, pp. 95-124).
The cellular iron uptake and transport is normally driven by a TF/TF receptor (TFR)-mediated endocytotic process (Baker, et al., Proc Natl Acad Sci USA 100: 3579-3583 (2003)). When TF is free of iron (apo-TF), both its N- and C-lobes adopt an open conformation through keeping two domains in each lobe well separated for easy access of the ferric iron. At the extracellular pH of 7.4, the apo-TF binds one (monoferric TF) or two iron molecules (diferric TF or holo-TF) by the coordination of iron-binding ligands. The diferric TF then binds to TFR on the cell surface in a way that the TF C-lobe binds laterally at the helical domain of dimeric TFR while the TF N-lobe is sandwiched between the TFR ectodomain and the cell membrane (Cheng, et al., Cell 116: 565-76 (2004); Cheng, et al., J. Struct. Biol. 152: 204-210 (2005)). This TF-TFR complex is then endocytosed into the early endosome, where the acidic environment (pH 5.5) triggers the conformational change of TF-TFR and the subsequent release of iron from TF by first protonating and dissociating the synergistic anion followed by protonating iron binding-related His and/or Tyr ligands (Baker, et al., Proc Natl Acad Sci USA 100: 3579-3583 (2003); Q.-Y. He, et al., “Molecular aspects of release of iron from transferrin,” in: D. M. Templeton, (Ed.), Molecular and Cellular Iron Transport, CRC Press, 2002, pp. 95-124). Finally, the apo-TF-TFR complex is recycled to the cell surface, where the neutral extracellular pH will dissociate the complex and release the TF for re-use.
The TF-TFR complex-mediated endocytosis pathway for iron transport is not only biologically significant for maintaining cellular iron homeostasis, but also has important pharmaceutical applications. TF is also an important ingredient of serum-free cell culture media due to its role in regulating cellular iron uptake, transport, and utilization in cultured cells. TF in serum-free cell culture medium ensures iron delivery to propagating cells for sustained growth in mammalian culture for the production of therapeutic proteins and vaccines (Barnes, et al., Cell 22: 649-55 (1980); Laskey, et al., Exp. Cell Res. 176: 87-95 (1988); Mortellaro, et al., Biopharm. International 20 (Supp) 30-37 (2007); Sharath, et al., J Lab Clin Med 103: 739-48 (1984)).
In addition, TF has also been actively pursued as a drug-delivery vehicle. As a drug carrier, TF increases a drug's therapeutic index via its unique transferrin receptor-mediated endocytosis pathway, as well as its added advantages of being biodegradable, nontoxic, and nonimmunogenic (Qian, et al., Med. Res. Rev. 22: 225-50 (2002); Qian, et al., Pharmacol. Rev. 54: 561-87 (2002); Soni, et al., American Journal of Drug Delivery 3: 155-70 (2005)). TF not only can deliver anti-cancer drugs to primary proliferating malignant cells where the TF is abundantly expressed (Qian, et al., Pharmacol. Rev. 54: 561-87 (2002)), but also can deliver drugs to the brain by crossing the blood-brain barrier (BBB), which is a major barrier for administrating sufficient drugs to reach the central nervous system (CNS) (Qian, et al., Med. Res. Rev. 22: 225-50 (2002); Soni, et al., American Journal of Drug Delivery 3: 155-70 (2005); Pardridge, Discov. Med. 6:139-43(2006)). TF can also be exploited for oral delivery of protein-based therapeutics (Bai, et al., Proc. Natl. Acad. Sci. U.S.A. 102: 7292-6 (2005); Widera, et al., Adv. Drug Deliv. Rev. 55:1439-66(2003)), as TF is resistant to proteolytic degradation and TFR is abundantly expressed in human gastrointestinal (GI) epithelium (Bai, et al., Proc. Natl. Acad. Sci. U.S.A. 102: 7292-6 (2005); Banerjee, et al., Gastroenterology 91: 861-9 (1986)).
With the increasing concerns over the risk of transmission of infectious pathogenic agents from the use of human or animal plasma-derived TFs in both cell culture and drug delivery applications, recombinant transferrin (rTF) is preferred to native TF (Keenan, et al., Cytotechnology 51: 29-37(2006)). Recombinant human TF (rhTF) has long been pursued in a variety of expression systems (MacGillivray, et al., “Transferrins” in: D. M. Templeton, (Ed.), Molecular and cellular iron transport, Marcel Dekker, New York, 2002, pp. 41-70), but proves to be challenging largely due to hTF's complicated structural characteristics as described above. The commonly used E. coli system for production of recombinant proteins has proved to be impractical for producing rhTF, as the expressed rhTF protein remains in insoluble inclusion bodies and the yield of functionally active rhTF after renaturation is very limited (Hoefkens, et al., Int. J. Biochem. Cell Biol. 28: 975-82 (1996)). Although both the insect cell (baculovirus) (Ali, et al., Biochem. J. 319 (Pt 1):191-5 (1996)) and mammalian cell (MacGillivray, et al., “Transferrins” in: D. M. Templeton, (Ed.), Molecular and cellular iron transport, Marcel Dekker, New York, 2002, pp. 41-70) expression systems have been shown to be able to express the bioactive rhTF, neither of them express at high enough levels to provide enough quantity to be a feasible source of commercial production, as well as being cost prohibitive.
It is shown herein that when transferrin is expressed in bacterial, yeast, mammalian cells, and insect cell expression systems, the expressed native transferrin protein bears a glycosylation pattern characteristic of the host organism, i.e., animal cell-expressed transferrin has a animal-type glycosylation pattern, and yeast-expressed transferrin has a yeast-type glycosylation pattern. It is desirable to produce a biologically active transferrin protein that is non-glycosylated for therapeutic use, to avoid possible allergic or immunological reactivity. Recently, bioactive rhTF was expressed in Saccharomyces cerevisiae using a mutated transferrin gene in which two of its N-linked glycosylation sites have been knocked out, and this rhTF became commercially available. (Sargent, et al., BioMetals (2006) 19:513-519). However, this yeast-derived rhTF, still remains very expensive to produce (Millipore, Billerica, Mass.). To address the problems of the shortage and the high cost of producing rhTF, as well as to meet a previously unmet need for producing high levels of an non-glycosylated human transferrin, alternative expression systems are desirable.
With the advancement of plant molecular biology in general and the improvement of plant transformation techniques in particular, plant hosts have become a powerful system to produce recombinant proteins cost-effectively and on a large scale (Daniell, et al., Trends Plant Sci. 6: 219-26 (2001); Lienard, et al., Biotechnol. Annu. Rev. 13: 115-47 (2007); Twyman, et al., Expert Opin. Emerg. Drugs 10: 185-218 (2005); Huang, et al., “ExpressTec: high level expression of biopharmaceuticals in cereal grains” in: K. J, (Ed.), Modern Biopharmaceuticals, Wiley VCH, 2005, pp. 931-47).
None of the aforementioned patents or publications discloses the production of non-glycosylated native transferrin protein in monocot seeds in high yield. It is desirable to provide for the production of non-glycosylated native transferrin protein in high yield free from contaminating source agents in order to provide a sufficient supply of transferrin in serum-free cell culture medium as well as in therapeutic compositions for the patient population with conditions treatable by administration of transferrin protein.